Stem cells are defined as cells that, at the single cell level, are capable of both self-renewal and differentiation to specialized cell types. The growth potential of mammalian embryonic stage cells have been known for many years, although the ability to culture such pluripotent and totipotent stem cells, particularly human stem cells, has only been recently developed. Embryonic stem (ES) cells are derived from cultures of inner cell mass (ICM) cells, and have the property of participating as totipotent cells when placed into host blastocysts. The developmental pathways that endogenous ICM cells or transferred ES cells take to tissue formation and organogenesis may be controlled for the development of tissue and organ specific stem cells. The ability of ES cells to grow specialized cells and tissues could provide an unprecedented tool in the clinic, by providing a means for transplantation and repair of damaged muscles, nerves, organs, bones and other tissues. ES cell lines also have a potent ability to replicate in culture, unlike many of the somatic stem cells, which may also be limited to differentiation within specific lineages.
When cultured in vitro, ES cells can self-renew indefinitely in the presence of LIF and fetal bovine serum or mouse feeder layer cells, resulting in daughter cells that maintain their potential for multilineage differentiation. In general, when ES cells are maintained in serum- and feeder-free conditions, the number of undifferentiated cells quickly reaches a plateau and begins to decline after only a couple of passages, and a population of cells with a non-ES cell morphology arises in culture despite the presence of LIF. Thus, additional signals from serum or feeders appear to be required to fully support the self-renewal of ES cells.
Serum and feeder cells may act to provide the survival signals manifest in growth factors and cytokines, and such extrinsic survival signals can be especially critical in low cell density conditions. Where such stimulation through auto- and paracrine factors is inadequate, ES cells may become apoptotic.
It has been suggested that the use of N2 and B27 supplemented media to expand ES cells in serum- and feeder-free conditions improves viability. However, these supplements alone cannot support the self-renewal of ES cells. Further, as N2 and B27 supplements contain hormones (corticosterone, progesterone, and T3) and retinyl acetate, a precursor of retinoic acid, (which components may induce differentiation of ES cells), the presence of these components complicates the analysis of cytokines, growth factors, and chemicals on self-renewal and differentiation of ES cells. To analyze the effect of single cytokines, growth factors, and other molecules on self-renewal and differentiation of ES cells, it would optimal if cells could be protected from apoptotic cell death in serum- and feeder-free conditions.
The therapeutic use of stem cells will benefit from the ability to culture the cells in defined media. Such media may have the advantage of being free from feeder layer cells, which have a potential for virus contamination. Such media may also be free of serum, which adds cost, complexity, and a potential for prion contamination. The present invention addresses this problem.